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Regulation of α2B‐AR expression in an in vitro system
Author(s) -
Duling Laura C,
Cherng Tom W,
Kanagy Nancy L
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1184-c
Subject(s) - enos , phenylephrine , mesenteric arteries , transfection , nitric oxide synthase , chemistry , medicine , endocrinology , lipofectamine , in vitro , nitric oxide , blood pressure , artery , biochemistry , gene , vector (molecular biology) , recombinant dna
Previously we reported that mice heterozygous (HET) for a non‐functional α 2B ‐adrenergic receptor (α 2B ‐AR) develop more hypertension then wild type (WT) mice when treated chronically with a nitric oxide synthase (NOS) inhibitor. We also reported that contractile responses to phenylephrine (PE) are decreased in arteries from HET but acute NOS inhibition restores PE constriction to that seen in WT arteries. Thus loss of α 2B ‐AR expression increases NO contribution to blood pressure and vascular tone. Additionally, Western analysis showed that mesenteric arteries from HET mice express more endothelial NOS (eNOS) than WT arteries. We hypothesized that reduced α 2B ‐AR expression directly regulates eNOS expression in endothelial cells. To determine if we could acutely decrease expression of α 2B ‐AR, we transfected NG108‐15 cells (neuroblastoma cells which constitutively express α 2B ‐AR) with an siRNA against α 2B ‐AR (Dharmacon). Using Lipofectamine 2000, cells were transfected with the siRNA for 24, 48, or 72 hours and then harvested. Western analysis showed that there was a slight decrease in expression at 48 hrs (22±4% decrease, p = 0.14) and a larger reduction after 72 hours of exposure (61±9% decrease p=0.060). However, eNOS was not detected in NG108‐15 cells. Thus future studies will use this protocol to directly address our hypothesis in cultured endothelial cells which express both eNOS and α 2B ‐AR.